Targeting fumarate hydratase promotes mitochondrial RNA-mediated interferon production

Metabolic rewiring underlies macrophage effector functions, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show induction of an inflammatory aspartate-argininosuccinate shunt following LPS stimulation. The shunt, supported by increased ASS1 expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacologic inhibition and genetic ablation of the TCA cycle enzyme FH further elevates intracellular fumarate levels, suppresses mitochondrial respiration, and increases mitochondrial membrane potential. RNA sequencing and proteomic analysis demonstrates profound inflammatory effects resulting from FH inhibition. Of note, acute FH inhibition suppresses IL-10 expression leading to increased TNF-α secretion, an effect recapitulated by fumarate esters. Unexpectedly, FH inhibition, but not fumarate esters, also increases IFN-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7 and RIG-I/MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged LPS stimulation. Furthermore, cells from SLE patients also exhibit FH suppression, indicating a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.


LPS stimulation drives an aspartate-argin inosuccinate shunt
To evaluate metabolic alterations that occur during LPS stimulation, we employed an unbiased liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach to characterise the metabolome of inflammatory bone marrow-derived macrophages (BMDMs).The TCA cycle metabolite fumarate stood out as one of the most significantly upregulated metabolites upon exposure to acute LPS stimulation, joining previously identified metabolites such as itaconate2 (Fig. 1a).We also observed a significant increase in fumarate-mediated protein succination6-8, resulting in the formation of the fumaratecysteine adduct, (S)-2-succinocysteine (2SC) (Extended Data Fig. 1a-c).
As acute LPS stimulation failed to impair respiration (Fig. 1b, c), TCA cycle disruption is unlikely to be sufficient for fumarate accumulation.Increased flux through the aspartateargininosuccinate shunt has been reported to support nitric oxide (NO) production3.As fumarate is a by-product of argininosuccinate cleavage by argininosuccinate lyase (ASL) in the cytosol, we hypothesised that argininosuccinate may be a source of fumarate.Supporting this, we observed decreased aspartate, the substrate for argininosuccinate, and increased argininosuccinate, fumarate, and malate levels (Fig. 1d), consistent with increased flux through the shunt.This rewiring also occurred during prolonged LPS stimulation (Extended Data Fig. 1d).
Inhibition of the aspartate-argininosuccinate shunt with the GOT2 inhibitor aminooxyacetic acid (AOAA)3 reduced aspartate, asparagine, argininosuccinate, fumarate and 2SC levels following LPS stimulation (Fig. 1h and Extended Data Fig. 1e, f).Knockdown of Asl also prevented fumarate accumulation (Extended Data Fig. 1g, h) indicating its dependency on the aspartate-argininosuccinate shunt, which would increase cytosolic fumarate (Fig. 1i).With stable isotope-assisted tracing, we show that glutamine-dependent anaplerosis is in part responsible for fumarate accumulation and drives the aspartate-argininosuccinate shunt.U-13C-glutamine tracing demonstrated glutaminolysis as a carbon source for the TCA cycle, aspartate-argininosuccinate shunt metabolites, including fumarate, and glutathione (Extended Data Fig. 2).15N2-glutamine tracing also demonstrated that glutamine nitrogen is a source for glutathione synthesis and aspartate-argininosuccinate shunt metabolites (Extended Data Fig. 3).Importantly, AOAA completely prevented the contribution of glutamine nitrogen to aspartate, asparagine, arginine and citrulline, confirming its inhibition of GOT2.Metabolomics on cytosolic fractions of resting and LPS-stimulated macrophages showed that metabolites such as itaconate and succinate accumulate in the cytosol following LPS stimulation (Extended Data Fig. 4a).Importantly, we also found increased cytosolic argininosuccinate, fumarate and 2SC (Extended Data Fig. 4b).

FH inhibition causes bioenergetic and redox stress
FH catalyses the hydration of fumarate to malate in the mitochondrion and cytosol11, the inhibition of which elevates cytosolic fumarate accumulation, perturbs urea cycle metabolism and leads to renal cyst development12.Given protein levels of FH remain stable during early LPS stimulation (Fig. 1g), we used a well-established pharmacological inhibitor of FH (FHIN1)13 and a recently developed tamoxifen-inducible CRE-ERT2-Fh1-""""""""""""""""-model to probe the role of FH activity and fumarate accumulation in macrophages.However, since FH inhibition may lead to effects independent of fumarate accumulation through mitochondrial and redox stress14, we also used low concentrations of cell-permeable dimethyl fumarate (DMF) to deliver a cysteine-reactive fumarate ester which does not inhibit respiration15-17.This approach would uncouple the role of impaired mitochondrial bioenergetics following TCA cycle disruption and fumaratemediated electrophilic modification of cysteine residues.

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Previous reports show that immunometabolites and their derivatives affect macrophage function through regulation of metabolic pathways7,18,19.We therefore aimed to assess how FH inhibition and DMF may regulate macrophage metabolism.First, comparing the effects of FHIN1 and DMF on mitochondrial bioenergetics, we found that FHIN1 reduced ratios of ATP/ADP, ATP/AMP, and P-creatine/creatine while DMF had no effect, demonstrating that FH sustains mitochondrial bioenergetics (Fig. 2a, Extended Data Fig. 5a).This was confirmed by respirometry, showing FHIN1 impaired basal respiration, ATP production and maximal respiration as measured by OCR, while DMF had no effect (Fig. 2b).FHIN1 led to a distinct metabolic signature characterised by alterations in TCA cycle metabolites including citrate, aconitate, itaconate and succinate, indicating TCA cycle rewiring, as well as enhanced fumarate and 2SC accumulation, supporting this approach in studying the roles of FH in macrophages (Fig. 2c, e, Extended Data Fig. 5b).Principal component analysis (PCA) showed a significant divergence of FHIN1 treatment to the other conditions (Fig. 2d).
As FHIN1 impaired respiration, we examined further mitochondrial parameters.We first observed increased reactive oxygen species (ROS) production in cells treated with FHIN1 but not DMF (Fig. 2g).FHIN1 treatment also increased staining intensity of the mitochondrial membrane potential (MMP)-dependent dye mitotracker RED (mtRED) (Extended Data Fig. 5j, k).Tetramethylrhodamine methyl ester (TMRM) staining confirmed this result, as FHIN1 significantly increased staining while DMF had no effect (Fig. 2h).Similarly, Fh1-/-macrophages had increased MMP, as previously reported in kidney epithelial cells22 (Fig. 2h).We also observed a decreased aconitate/citrate ratio in FHIN1treated macrophages, indicative of impairment in the fumarate-and redox-sensitive TCA cycle enzyme aconitase23 (Fig. 2i).Although the GSSG/GSH ratio was unchanged, FHIN1 led to a depletion of total glutathione (Fig. 2j), consistent with fumarate-mediated glutathione depletion24,25.These data suggest that FH inhibition induces profound redox stress responses.

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FH activity is required to maintain appropriate cytokine responses
To determine whether FH regulates macrophage activation and effector responses, we performed RNA sequencing and proteomics to assess changes in the transcriptome and proteome of FHIN1-treated BMDMs.Geneset enrichment analysis (GSEA) identified an expected suppression in genes associated with metabolism, but FHIN1 also decreased expression of inflammatory pathways, including IL-1 and IL-10 signalling (Fig. 3a).Increased expression of the heme-regulated inhibitor (HRI) stress response, amino acid metabolism and tRNA aminoacylation was also observed (Fig. 3a), consistent with previous reports14.Further overrepresentation analysis (ORA) of RNAseq data revealed TNF-α signalling to be the most highly upregulated pathway in our analysis (Fig. 3b).
Comparing FHIN1 with DMF on cytokine readouts allowed us to determine the role of protein succination following FH inhibition.Validating our transcriptomic analysis, FHIN1 and DMF decreased IL-10 release and expression, while TNF-α release and expression were increased (Fig. 3c, Extended Data Fig. 6a).Both compounds also reduced IL-1β expression and IL-6 release (Extended Data Fig. 6b), consistent with previous reports8,26, demonstrating widespread regulation of cytokine expression.
The less electrophilic fumarate ester, monomethyl fumarate (MMF), exhibited the same effects on Il10 and Tnfa expression (Fig. 3d), supporting a role for their regulation by fumarate.Shared transcriptomic changes of FHIN1 and DMF demonstrated strong downregulation of the ERK1/2 cascade and PI3K signalling (Fig. 3e).A similar transcriptional fingerprint has been observed in FH-deficient leiomyomas27.We also observed increased amino acid metabolism and transport, and autophagy transcripts (Extended Data Fig. 6c).Upon LPS stimulation, IL-10 is regulated by ERK1/2 and PI3Kinduced AP-1 activation28, suggesting that downregulation of this signalling axis by FHIN1 and DMF may repress IL-10.However, we did not observe changes in the upstream kinases (AKT, JNK, ERK and p38) which converge on AP-1 activation, (Extended Data Fig. 6d).Although we did observe reduced Jun expression in our transcriptomics dataset (Extended Data Fig. 6e), this could indicate reduced autoregulation by AP-129.In this dataset, Fos was not reduced (Extended Data Fig. 6f).
Interestingly, the thiol precursor N-acetyl cysteine (NAC) abrogated the suppression of Il10 by FHIN1 and DMF (Fig. 3f).The free thiols of NAC and its products would react with and sequester fumarate, thereby reducing the modification of protein thiols and suggesting that suppression of IL-10 results from a redox-dependent succination event.The electrophile sulforaphane has been shown to reduce AP-1 activation via modification of Cys-154 on c-Fos30.We therefore investigated if FHIN1 or DMF may affect c-Fos activation, despite upstream regulators remaining unaffected.Using a c-Fos transcription factor assay, we found that FHIN1 and DMF strongly impaired c-Fos activation (Fig. 3g), providing evidence of direct regulation of c-Fos, potentially through S-alkylation.IL-10 signalling has been shown to repress TNF-α expression3l.We confirmed this using an IL-10 receptor (CD210) blocking antibody targeting IL-10-mediated STAT3 phosphorylation, leading to augmented LPS-induced TNF-α release (Fig. 3h, Extended Data Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Fig. 6g).We then examined whether recombinant IL-10 supplementation could rescue the increase in TNF-α.Indeed, with IL-10, FHIN1 failed to impair STAT3 phosphorylation or augment TNF-α production (Fig. 3i, j), indicating that the FHIN1-and DMF-driven induction of TNF-α is dependent on the suppression of IL-10.
FH inhibition also resulted in the activation of an NRF2 and ATF4 stress response in macrophages (Extended Data Fig. 7a), in line with previous observations in epithelial cells14.Proteomic analysis revealed that the inflammation-associated hormone GDF1532-34 is one of the most significantly increased proteins with FHIN1 and DMF, while FHIN1 also increased the recently identified mitochondrial glutathione importer, SLC25A3935, reinforcing the mitochondrial redox perturbation (Extended Data Fig. 7b,  c).Validating our proteomics data, FH inhibition drove GDF15 release from macrophages (Extended Data Fig. 7d).Both ATF4 and NRF2 have been reported to regulate GDF15 in different contexts33,36, and silencing of each revealed that FHIN1-driven GDF15 release was partly NRF2-but not ATF4-dependent (Extended Data Fig. 7e, f).This work defines two previously unappreciated signalling axes linked to FH inhibition, uncovering its role in the regulation of IL-10/TNF-α and GDF15.The recent developments identifying GDF15 as a mediator of immune tolerance, and the anti-inflammatory properties of colchicine and NSAIDS36,37, suggest that protective effects of DMF in models of inflammation could be via GDF15.Additionally, increased TNF-α levels potentially explain adverse events reported with fumarate esters38.Mechanistically, suppression of IL-10 may also explain why fumarate esters promote enhanced TNF-α production during trained immunity, in addition to reported epigenetic changes39.
Strikingly, FHIN1, but not DMF or MMF, was found to increase IFN-13 release from LPS-stimulated macrophages (Fig. 4b, c).This was independent of NAC-sensitive redox stress (Extended Data Fig. 8e), and was not due to augmented TLR4 signalling, as LPSinduced TRAF3 levels and IL-1b expression were not increased by FHIN1 (Extended Data Fig. 8f, h).FHIN1 and DMF did modestly augment LPS-induced p65 phosphorylation (Extended Data Fig. 8g), which may contribute to increased TNF-a release41.Given FH inhibition causes mitochondrial stress (Fig. 2) which is associated with the release of immunostimulatory mitochondrial nucleic acids42-44, we hypothesised that the IFN response was driven by cytosolic nucleic acid sensors, such as cGAS.To support this, FH deficient-hereditary leiomyomatosis and renal cell cancer (HLRCC) tumours exhibit changes in mitochondrial DNA (mtDNA)20.We first used ethidium bromide (EtBr) to deplete mtDNA45 (Extended Data Fig. 8i) before treating cells with FHIN1 and LPS.We found that FHIN1 no longer boosted LPS-induced IFN-13 release in the presence of EtBr (Fig. 4d), indicating that increased IFN-13 release with FHIN1 may be mtDNA-dependent.We subsequently found that FHIN1 caused an increase in both mtDNA and mtRNA in cytosolic extracts (Fig. 4e, Extended Data Fig. 8j).Given the established role of mtDNA in driving IFN responses42,43, we examined whether the cGAS-STING or TLR9 DNAsensing pathways were required for the increase in IFN-13.However, neither use of the STING inhibitor C-17846 nor silencing of Cgas (cGAS) or Tmem173 (STING) had any effect on FHIN1-driven IFN-13 induction (Extended Data Fig. 8k-n).Targeting TLR9 using the competitive inhibitor ODN 208847 or using siRNA also had no effect on this response (Extended Data Fig. 8k-n).Suppression of Tmem173 expression by FHIN1 and DMF (Extended Data Fig. 8o) may explain why cGAS-STING signalling is redundant in our model, even in the presence of cytosolic mtDNA.ETC inhibition, as we observe with FHIN1 treatment, has also been shown to inhibit STING activation48.
Since cytosolic mtRNA was also increased by FHIN1 (Fig. 4e), we performed immunofluorescence staining with an antibody specific for double-stranded RNA (dsRNA).Mitochondrial RNA has previously been shown to drive an IFN response in human cells49,50, and is known to be particularly immunostimulatory51.FHIN1 treatment led to an accumulation of dsRNA relative to DMSO control (Fig. 4f).We subsequently co-treated cells with FHIN1 and IMT1, the mitochondrial RNA polymerase (POLRMT) inhibitor.The increase in mtRNA with FHIN1 was observed in the cytosolic fraction but not in the whole cell fraction and was inhibited in both by co-treatment with IMT1 (Extended Data Fig. 8p, q).Importantly, IMT1 also abrogated the FHIN1-mediated boost in IFN-13 release (Extended Data Fig. 8r), implicating the role of mtRNA in driving this response.Mitochondrial ssRNA, resulting from a decline in mitochondrial integrity, has also been Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts implicated in driving TLR7-dependent IFN signalling52,53.We subsequently silenced Tlr7 or the dsRNA sensors Ddx58 (RIG-I) and Ifih1 (MDA5) (Extended Data Fig. 9a, b), all of which abrogated the boost in IFN-13 release observed with FH inhibition (Fig. 4g, h), confirming a non-redundant requirement of these sensors and mtRNA, rather than mtDNA, for the FHIN1-driven IFN response.Knockdown of the cell surface dsRNA sensor Tlr3 did not affect the augmentation in IFN-b release (Extended Data Fig. 9c).RIG-I and MDA5, although predominantly described as dsRNA sensors, can also bind ssRNA54, indicating that the IFN response following FH inhibition is likely driven by a mixture of dsRNA and ssRNA species.It is notable that FHIN1 also reduced Ddx58 but not Ifih1 expression, which may warrant further investigation (Extended Data Fig. 9b).The signalling events downstream of RIG-I/MDA5 activation include mitochondrial antiviral signalling protein (MAVS) oligomerisation, followed by recruitment and phosphorylation of TANK-binding kinase 1 (TBK1).We observed MAVS oligomerisation and increased TBK1 phosphorylation with FHIN1 treatment (Fig. 4i, Extended Data Fig. 9d).Intriguingly, MAVS knockout did not impair the induction of IFN-13 by FHIN1 (Extended Data Fig. 9e), perhaps indicating that compensatory TLR7 signalling is sufficient to drive type I IFN following FH inhibition with chronic MAVS deficiency.
Previously, we demonstrated that FH inhibition causes mitochondrial stress (Fig. 2).Changes in MMP have previously been correlated with increased type I IFN release55, thus we hypothesised that disturbances in MMP may be linked to mtRNA release and IFN-13 induction following FH inhibition.To support this, we induced changes in MMP by using the ATP synthase inhibitor oligomycin A, which boosted MMP, the K+ ionophore valinomycin A, which non-significantly reduced MMP, or the uncoupler CCCP, which significantly dissipated MMP (Extended Data Fig. 9f, h).All treatments boosted LPS-driven IFN-13 release, akin to FHIN1 (Extended Data Fig. 9g, h).MMF, which does not increase LPS-induced IFN-13 expression (Fig. 4c), did not affect MMP (Extended Data Fig. 9i).Oligomycin treatment led to an accumulation of dsRNA to a similar extent to that observed in cells treated with FHIN1 or transfected with dsRNA (poly (I:C)), and increased mtRNA release into the cytosol (Extended Data Fig. 9j-l).Valinomycin treatment similarly drove dsRNA accumulation (Extended Data Fig. 9m, n), indicating that MMP-altering compounds induce an accumulation of mtRNA.As we also observed an increase in cytosolic mtDNA levels following oligomycin treatment (Extended Data Fig. 9l), it is still possible that IFN responses following oligomycin/valinomycin/CCCP treatment are not exclusively driven by mtRNA.mtRNA release from chondrocytes has recently been implicated in activating the immune response and promoting osteo-arthritis56.As such, mitochondrial damage and nucleic acid release are emerging as key pathogenic processes that may underlie many immune-mediated diseases.

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We next considered whether this response could be applied to an endogenous model of LPS activation in the absence of pharmacological or genetic inactivation of FH.
Given LPS-induced FH suppression occurs predominantly during late-phase LPS stimulation (24-48 h) (Fig. 1g), FH suppression at this time point may drive membrane hyperpolarisation and the release of mtRNA.MMP was significantly increased following 48 h LPS stimulation, but not following 4 h or 24 h stimulation (Extended Data Fig. 10a).Although dsRNA did not accumulate following acute (4 h) LPS stimulation (Extended Data Fig. 9j, k), we did observe increased dsRNA staining following 24 h and 48 h LPS stimulation (Extended Data Fig. 10b, c).Ddx58 and Ifihl expression is LPS-inducible (Extended Data Fig. 10d), which may suggest that RIG-I/MDA5 signalling is required during LPS stimulation.Indeed, silencing of Ddx58 and Ifihl reduced both 24 h and 48 h LPS-induced Ifnb1 expression (Fig. 4l), indicating that Ifnb1 transcription during late-phase LPS stimulation is maintained by mtRNA release.These results demonstrate that the mitochondrial retrograde type I IFN response, which we initially unmasked by pharmacologically or genetically targeting FH during early LPS signalling, is active endogenously during late-phase LPS activation with potential implications for chronic inflammation, for example during ageing57.
To determine whether FH inhibition leads to similar effects in vivo, we injected mice with FHIN1 or DMF prior to administration of LPS, and measured IFN-13 release into the serum.FHIN1 increased LPS-induced IFN-13 release, while DMF had no effect (Fig. 4m), indicating that FH inhibition leads to a similar IFN response in vivo which may have effects on bystander cells.We also treated human PBMCs with FHIN1 or DMF prior to LPS stimulation and observed similar effects, as FHIN1 boosted, while DMF suppressed LPS-induced IFN-13 release (Fig. 4n).
We hereby describe a mitochondrial retrograde signalling pathway leading from FH inhibition to mitochondrial membrane hyperpolarisation and mtRNA release (Extended Data Fig. 11).Mitochondrial stress may be an underlying mechanism that contributes to type I IFN release in interferonopathies such as systemic lupus erythematosus (SLE).It has previously been demonstrated that PBMCs from SLE patients have impaired mitochondrial function and altered MMP58,59.We therefore examined FH expression in the whole blood of SLE patients and found significant suppression of FH compared to healthy control samples (Fig. 4o).Autoantibodies to dsRNA, as well as dsDNA, have been detected in SLE patients60,61.However, it is unclear whether FHs uppression is a cause or consequence of increased IFN signalling, as Fh1 can also be inhibited by IFN-β stimulation in BMDMs (Extended Data Fig. 10e).A negative feedback loop may exist whereby suppression of FH leads to type I IFN release, which feeds back to further suppress FH.FH suppression has previously also been linked to multiple sclerosis progression62 and, in parallel to our work, has been shown to promote a type I IFN response in kidney epithelial cells and HLRCC tumours (Zecchini, Paupe et al., under revision).This study and ours implicate roles for FH in nucleic acid release, which may contribute to inflammation-driven tumorigenesis and as a potential host defence mechanism in the context of viral infection.Finally, the recent demonstration of aberrant dsRNA editing due to ADAR1 deficiency leading to MDA5 activation as a mechanism of common inflammatory diseases also points to the clinical Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts relevance of endogenously produced dsRNA, suggesting that targeting this pathway may yield novel anti-inflammatory strategies63.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts

Fig. 1 .
LPS stimulation drives fumarate accumulation and protein succinationHooftman et al.Page 10 Nature.Author manuscript; available in PMC 2024 February 15.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 2. LPS stimulation drives fumarate accumulation via glutamine anaplerosis and an aspartate-argininosuccinate shunt Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 3. LPS stimulation drives fumarate accumulation via glutamine anaplerosis and an aspartate-argininosuccinate shunt Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 4. Increase in aspartate-argininosuccinate shunt metabolites in cytosol and Irg1-KO macrophages Extended Data Fig. 5. FH deletion increases bioenergetic stress, fumarate and mitochondrial membrane potential Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 6.FH inhibition remodels inflammatory gene expression Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 7. FH inhibition triggers the NRF2 and ATF4 stress response and promotes GDF15 release Extended Data Fig. 8. IFN-β release following FH inhibition is independent of cGAS-STING signalling Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Extended Data Fig. 9. Mitochondrial membrane potential modifiers increase mtRNAand trigger IFNβ release Extended Data Fig.